Engineered enzymes with methionine-gamma-lyase enzymes and pharmacological preparations thereof

ABSTRACT

Methods and composition related to the engineering of a novel protein with methionine-γ-lyase enzyme activity are described. For example, in certain aspects there may be disclosed a modified cystathionine-γ-lyase (CGL) comprising one or more amino acid substitutions and capable of degrading methionine. Furthermore, certain aspects of the invention provide compositions and methods for the treatment of cancer with methionine depletion using the disclosed proteins or nucleic acids.

This application is a divisional of co-pending U.S. patent applicationSer. No. 14/225,518, filed Mar. 26, 2014, which is a divisional of U.S.patent application Ser. No. 13/020,268, filed Mar. 2, 2011, now U.S.Pat. No. 8,709,407, which claims priority to U.S. Provisional PatentApplication No. 61/301,368 filed on Feb. 4, 2010. The entire contents ofeach of the above referenced disclosures are specifically incorporatedherein by reference.

This invention was made with government support under Grant no. R01CA139059 and R01 CA154754 awarded by the National Institutes of Health.The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The sequence listing that is contained in the file named“GGEOP0004USD2_ST25.txt”, which is 45 KB (as measured in MicrosoftWindows®) and was created on Feb. 10, 2016, is filed herewith byelectronic submission and is incorporated by reference herein.

1. Field of the Invention

The invention generally relates to compositions and methods for thetreatment of cancer with enzymes that deplete L-Methionine. Moreparticularly, it concerns the engineering of a novel humanmethionine-γ-lyase enzyme with methionine degrading activity and greatlyenhanced stability suitable for human therapy.

2. Description of Related Art

The demand for the essential amino acid, methionine, is exceptionallyhigh in cancerous tissues. Depletion of methionine has been shown to beeffective in killing a wide variety of tumor types without adverselyeffecting non-cancerous tissues. Methionine depletion can be effectedvia the action of enzymes that hydrolyze the amino acid. While humanmethionine depleting enzymes do not exist, a bacterial enzyme fromPseudomonas aeruginosa, methionine-γ-lyase, has been shown to betherapeutically effective in the clinic and has been evaluated inclinical trials. However, methionine-γ-lyase, as a bacterial protein, ishighly immunogenic and elicits the formation of specific antibodies,leading to adverse reactions and reduced activity. Methionine-γ-lyasealso has a very short half-life of only ˜2 hrs in vitro and in vivo,necessitating very frequent and impractically high dosing to achievesystemic depletion.

Systemic methionine depletion is the focus of much research and has thepotential to treat cancers such as metastatic breast cancer, prostate,neuroblastoma, and pancreatic carcinoma among others. Although there isa lot of excitement for this therapeutic approach, the bacteriallyderived methionine-γ-lyase has serious shortcoming which greatly dampensenthusiasm for it as a chemotherapeutic agent.

Thus, there remains a need to develop methods and compositions toaddress these shortcomings for the therapeutic success of L-methioninedepletion therapy.

SUMMARY OF THE INVENTION

Certain aspects of the present invention overcome a major deficiency inthe art by providing novel enzymes that comprise human polypeptidesequences having methionine-γ-lyase (MGL) activity, which may besuitable for cancer therapy and have low immunogenicity and improvedserum stability. Accordingly, in a first embodiment there is provided amodified polypeptide, particularly a novel enzyme variant withmethionine degrading activity derived from primate enzymes related toMGL. For example, the novel enzyme variant may have an amino acidsequence selected from the group consisting of SEQ ID NO: 10-17. Inparticular, the variant may be derived from human enzymes such as humancystathionine-γ-lyase (CGL). In certain aspects, there may be apolypeptide comprising a modified human cystathionine gamma-lyasecapable of degrading methionine. In some embodiments, the polypeptidemay be capable of degrading methionine under physiological conditions.For example, the polypeptide may have a catalytic efficiency formethionine (k_(cat)/K_(M)) of at least or about 0.01, 0.05, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10⁴, 10⁵, 10⁶ M⁻¹s⁻¹ or any range derivable therein. In further aspects, the polypeptidemay display a catalytic activity towards L-homocystine up tok_(cat)/K_(M) of 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40,35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6,0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001 M⁻¹ s⁻¹ or any rangederivable therein.

A modified polypeptide as discussed above may be characterized as havinga certain percentage of identity as compared to an unmodifiedpolypeptide (e.g., a native polypeptide) or to any polypeptide sequencedisclosed herein. For example, the unmodified polypeptide may compriseat least or up to about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150,200, 250, 300, 350, 400 residues (or any range derivable therein) of anative primate cystathionase (i.e., cystathionine gamma lyase). Thepercentage identity may be about, at most or at least 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or anyrange derivable therein) between the modified and unmodifiedpolypeptides, or between any two sequences in comparison. It is alsocontemplated that percentage of identity discussed above may relate to aparticular modified region of a polypeptide as compared to an unmodifiedregion of a polypeptide. For instance, a polypeptide may contain amodified or mutant substrate recognition site of cystathionase that canbe characterized based on the identity of the amino acid sequence of themodified or mutant substrate recognition site of cystathionase to thatof an unmodified or mutant cystathionase from the same species or acrossthe species. A modified or mutant human polypeptide characterized, forexample, as having at least 90% identity to an unmodified cystathionasemeans that 90% of the amino acids in that modified or mutant humanpolypeptide are identical to the amino acids in the unmodifiedpolypeptide.

Such an unmodified polypeptide may be a native cystathionase,particularly a human isoform or other primate isoforms. For example, thenative human cystathionase may have the sequence of SEQ ID NO: 1.Non-limiting examples of other native primate cystathionases includePongo abelii cystathionase (Genbank ID: NP_001124635.1; SEQ ID NO:18),Macaca fascicularis cystathionase (Genbank ID: AAW71993.1; SEQ IDNO:19), and Pan troglodytes cystathionase (Genbank ID: XP_513486.2; SEQID NO: 20). Exemplary native polypeptides include a sequence havingabout, at most or at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identity (or any range derivabletherein) of SEQ ID NO:1 or 18-20 or a fragment thereof. For example, thenative polypeptide may comprise at least or up to about 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 415 residues (orany range derivable therein) of the sequence of SEQ ID NO:1 or 18-20.

In some embodiments, the native cystathionine gamma-lyase may bemodified by one or more other modifications such as chemicalmodifications, substitutions, insertions, deletions, and/or truncations.For example, the modifications may be at a substrate recognitions siteof the native enzyme. In a particular embodiment, the nativecystathionine may be modified by substitutions. For example, the numberof substitutions may be one, two, three, four or more. In furtherembodiments, the native cystathionine gamma-lyase may be modified in thesubstrate recognition site or any location that may affect substratespecificity. Particularly, the amino acids that may be modified arecharged residues, such as negatively charged (e.g., Asp, Asn) orpositive charged residues (e.g., Arg, Lys). For example, the modifiedpolypeptide may have the at least one amino acid substitution at aminoacid positions corresponding to E59, E119 and/or R339 of SEQ ID NO:1 oramino acid positions of 59, 119 and/or 339 of a primate cystathioninegamma-lyase. For example, the primate may be human, Pongo abelii, Macacafascicularis, Pan Troglodyte.

In certain embodiments, the modifications may be substitutions of one ormore charged residues (including positively charged residues such as Argor R, Lys or K, His or H and negatively charged residues such as Asp orD and Glu or E) with neutral residues (e.g., Ala, Asn, Gln, Gly, Ile,Leu, or Val). For example, the substitutions at amino acid positions 59,119 and/or 339 is an aspartic acid (N), a valine (V), or a leucine (L).In particular embodiments, the modification are one or moresubstitutions selected from the group consisting of E59N, E59V, R119L,and E339V. In a further embodiment, the substitutions may comprise aR119L and E339V substitutions. In a still further embodiment, thesubstitutions may comprise an additional substitutions of E59N or E59V.

In some embodiments, the native cystathionine-γ-lyase may be a humancystathionine-γ-lyase. In a particular embodiment, the substitutions area combination of E59N, R119L, and E339V of human cystathioninegamma-lyase (for example, the modified polypeptide having the amino acidsequence of SEQ ID NO:10, a fragment or homolog thereof) or acombination of E59V, R119L, and E339V of human cystathionine gamma-lyase(for example, the modified polypeptide having the amino acid sequence ofSEQ ID NO:11, a fragment or homolog thereof). In a further embodiment,the modified polypeptide may be Pongo abelii Pongo abelii CGL-NLV mutant(SEQ ID NO:12), Pongo abelii CGL-VLV mutant (SEQ ID NO:13); Macacafascicularis CGL-NLV mutant (SEQ ID NO:14), Macaca fascicularis CGL-VLVmutant (SEQ ID NO:15); Pan Troglodytes CGL-NLV mutant (SEQ ID NO:16),and Pan Troglodytes CGL-VLV mutant (SEQ ID NO:17).

In some aspects, the present invention also contemplates polypeptidescomprising the modified cystathionine gamma-lyase linked to aheterologous amino acid sequence. For example, the modifiedcystathionine gamma-lyase may be linked to the heterologous amino acidsequence as a fusion protein. In a particular embodiment, the modifiedcystathionine gamma-lyase may be linked to an XTEN polypeptide forincreasing the in vivo half-life.

To increase serum stability, the modified cystathionine gamma-lyase maybe linked to one or more polyether molecules. In a particularembodiment, the polyether may be polyethylene glycol (PEG). The modifiedpolypeptide may be linked to PEG via its specific amino acid residues,such as lysine or cysteine. For therapeutical administration, such apolypeptide comprising the modified cystathionine gamma-lyase may bedispersed in a pharmaceutically acceptable carrier.

In some aspects, a nucleic acid encoding such a modified cystathioninegamma-lyase is contemplated. In some embodiments, the nucleic acid hasbeen codon optimized for expression in bacteria. In particularembodiments, the bacteria is E. coli. In other aspects, the presentinvention further contemplates vectors such as expression vectorscontaining such nucleic acids. In particular embodiments, the nucleicacid encoding the modified cystathionine gamma-lyase is operably linkedto a promoter, including but not limited to heterologous promoters.

In still further aspects, the present invention further contemplateshost cells comprising such vectors. The host cells may be bacteria, suchas E. coli To further differentiate desired CGL mutants with methioninedegrading activity from the native cystathionine gamma-lyase, host cellshaving deletions of ilvA and metA (e.g., E. coli ilvA-metA-) may beprepared and used to identify desired mutants.

In a further embodiment, there may be also provided a method of ofidentifying a primate cystathionine gamma-lyase variant havingL-methionine degrading activity, comprising: a) expressing a populationof primate cystathionine gamma-lyase variants in cells of an E. colistrain having deletions of genes ilvA and metA, wherein the variantcomprises at least one amino acid substitution as compared to a nativeprimate cystathionine gamma-lyase; and b) identifying a primatecystathionine gamma-lyase variant having L-methionine degradingactivity, wherein cells expressing the identified variant has a highergrowth rate in a minimal medium supplemented with L-methionine ascompared to cells expressing the native primate cystathioninegamma-lyase in otherwise identical conditions.

In some embodiments, the vectors are introduced into host cells forexpressing the modified cystathionine gamma lyase. The proteins may beexpressed in any suitable manner. In one embodiment, the proteins areexpressed in a host cell such that the protein is glycosylated. Inanother embodiment, the proteins are expressed in a host cell such thatthe protein is aglycosylated.

Certain aspects of the present invention also contemplate methods oftreatment by the administration of the modified cystathioninegamma-lyase peptide, the nucleic acid, or the formulation of the presentinvention, and in particular methods of treating tumor cells or subjectswith cancer. The subject may be any animal such as mouse. For example,the subject may be a mammal, particularly a primate, and moreparticularly a human patient. In some embodiments, the method maycomprise selecting a patient with cancer. In certain aspects, thesubject or patient may be maintained on a methionine-restricted diet ora normal diet.

In some embodiments, the cancer is any cancer that is sensitive tomethionine depletion. In one embodiment, the present inventioncontemplates a method of treating a tumor cell or a cancer patientcomprising administering a formulation comprising such a polypeptide. Insome embodiments, the administration occurs under conditions such thatat least a portion of the cells of the cancer are killed. In anotherembodiment, the formulation comprises such a modified cystathioninegamma-lyase with methionine degrading activity at physiologicalconditions and further comprising an attached polyethylene glycol chain.In some embodiment, the formulation is a pharmaceutical formulationcomprising any of the above discussed cystathionine gamma-lyase variantsand pharmaceutically acceptable excipients. Such pharmaceuticallyacceptable excipients are well known to those having skill in the art.All of the above cystathionine gamma-lyase variants may be contemplatedas useful for human therapy.

In a further embodiment, there may also be provided a method of treatinga tumor cell comprising administering a formulation comprising anon-bacterial (mammalian, e.g., primate or mouse) modified cystathioninegamma-lyase that has methionine degrading activity or a nucleic acidencoding thereof.

Because tumor cells are dependent upon their nutrient medium formethionine, the administration or treatment may be directed to thenutrient source for the cells, and not necessarily the cells themselves.Therefore, in an in vivo application, treating a tumor cell includescontacting the nutrient medium for a population of tumor cells with theengineered methioninase. In this embodiment, the medium can be blood,lymphatic fluid, spinal fluid and the like bodily fluid where methioninedepletion is desired.

In accordance with certain aspects of the present invention, such aformulation containing the modified cystathionine gamma-lyase can beadministered intravenously, intradermally, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostaticaly, intrapleurally, intrasynovially, intratracheally,intranasally, intravitreally, intravaginally, intrarectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,subconjunctival, intravesicularlly, mucosally, intrapericardially,intraumbilically, intraocularly, orally, topically, by inhalation,infusion, continuous infusion, localized perfusion, via a catheter, viaa lavage, in lipid compositions (e.g., liposomes), or by other method orany combination of the forgoing as would be known to one of ordinaryskill in the art.

Embodiments discussed in the context of methods and/or compositions ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the terms “encode” or “encoding” with reference to anucleic acid are used to make the invention readily understandable bythe skilled artisan; however, these terms may be used interchangeablywith “comprise” or “comprising” respectively.

As used herein in the specification, “a” or “an” may mean one or more.As used herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: CGL and MGL catalyze a similar PLP dependent degradation ofL-cystathionine or L-Methionine (L-Met) into ammonia, α-ketobutyrate,and cysteine or methanethiol respectively.

FIG. 2: Plot of % activity over time in pooled human serum incubated at37° C. MGL from p. putida (∘) with an apparent T_(0.5) of 2±0.1 hrs,human CGL (▴) with an apparent T_(0.5) of 16±0.8 hrs, variant hCGL-NLV(♦) with an apparent T_(0.5) of 95±3 hrs, and variant hCGL-VLV (•) withan apparent T_(0.5) of 260±18 hrs.

FIG. 3: Human CGL (▴) and variant hCGL-NLV (♦) with an apparent T_(M)values of ˜68° C., and variant hCGL-VLV (•) with an apparent T_(M) of˜71° C.

FIG. 4: PEGylation greatly increases the apparent molecular weight ofhCGL variants. SDS-PAGE of MW ladder (lane 1), purified hCGL variant(lane 2), and purified hCGL variant modified with varying amounts of PEGNETS-ester MW 5000 (10, 20, 40 & 80 fold molar excess, lanes 3-6respectively.

FIG. 5: PEGylation greatly increases the apparent molecular weight ofhCGL variants. SEC (size exclusion chromatography) chromatographyshowing an apparent MW of ˜1,340 kDa (A) for PEGylated hCGL variant and˜220 kDa for unpegylated hCGL variant (B).

FIG. 6: Effect of L-Met depleting enzymes on neuroblastoma cell lineLan-1. Recombinant pMGL (∘) with an apparent 1050 of 0.6 μM andrecombinant hCGL-NLV (♦) with an apparent IC₅₀ of 0.3 μM.

FIG. 7: Pharmacodynamic analysis of PEG-hCGL-NLV. The percentage ofenzyme activity in serum samples relative to t=0 is plotted. (♦) overtime with an apparent activity T½ of 28±4 hrs.

FIG. 8: A Met(−)Hcyss(−)Chl(−) diet was fed to athymic mice beforeadministration of 200 U PEG-hCGL-NLV (N=5). Serum methionineconcentration was expressed as mean±SD. Blood methionine level decreasedto a nadir of 3.9±0.7 μmol/L at 8 hours.

FIG. 9: Comparison of PEGylated hCGL-NLV with pMGL of the in vitroinhibition of proliferation of various neuroblastoma cell lines.

FIG. 10: Athymic mice bearing LAN-1 xenografts. (□) Control with normaldiet (N=10); (•) Met(−)Hcyss(−)Chl(−) mouse feed (N=10); (∘) 100 UPEG-hCGL-NLV in combination with Met(−)Hcyss(−)Chl(−) mouse feed (N=10)(▴ treatment days). Tumor growth rate was expressed as mean±SEM(standard error of the mean) for each group. * p<0.01 when the treatmentof PEG-hCGL-NLV in combination with Met(−)Hcyss(−)Chl(−) mouse feed wascompared with other two groups.

FIG. 11: Schematic of E. coli L-methionine and L-isoleucine syntheticpathways showing a double deletion of genes ilvA and metA. This rendersE. coli auxotrophic for L-Met and L-Ile. If E. coli are grown on mediasupplemented with L-Met and harbor a gene encoding an activemethionine-γ-lyase the resulting α-ketobutyrate production willcompensate for the L-Ile auxotrophy.

FIG. 12: E. coli BL21(DE3) (ΔilvA ΔmetA) plated on M9 minimal media agarsupplemented with 0.5 mM L-methionine and containing either a plasmidencoding a gene for methionine-γ-lyase (left) or a gene encoding acystathionine-γ-lyase (right). Only MGL activity is able to rescue theL-isoleucine auxotrophy.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present disclosure, according to certain embodiments, is generallydirected to compositions and methods for preparing a novel human enzymeengineered to have methionine degrading activity and its relatedtherapeutic applications.

Without wishing to be bound by theory or mechanism, the presentdisclosure is based on the following studies. A high throughput assaywas first developed for detecting methionine-γ-lyase by monitoring theformation of the product α-keto butyrate in 96 well plates and a secondassay was also developed to rapidly determine enzyme kinetics followingformation of methane thiol. A genetic selection for L-methionedegradation activity was devised, based on the formation ofα-ketobutyrate by the enzymes which in turn rescues the growth of E.coli ilvA mutant cells. Furthermore, saturation mutagenesis and randommutagenesis followed by high throughput screening for methioninedegradation were performed to isolate cystathionine-γ-lyase variantswith high methionine-γ-lyase activity.

As used herein the terms “protein” and “polypeptide” refer to compoundscomprising amino acids joined via peptide bonds and are usedinterchangeably.

As used herein, the term “fusion protein” refers to a chimeric proteincontaining proteins or protein fragments operably linked in a non-nativeway.

As used herein, the term “half life” (½-life) refers to the time thatwould be required for the concentration of a polypeptide thereof to fallby half in vitro or in vivo, for example, after injection in a mammal.

The terms “in operable combination,” “in operable order” and “operablylinked” refer to a linkage wherein the components so described are in arelationship permitting them to function in their intended manner, forexample, a linkage of nucleic acid sequences in such a manner that anucleic acid molecule capable of directing the transcription of a givengene and/or the synthesis of desired protein molecule, or a linkage ofamino acid sequences in such a manner so that a fusion protein isproduced.

By the term “linker” is meant to refer to a compound or moiety that actsas a molecular bridge to operably link two different molecules, whereinone portion of the linker is operably linked to a first molecule, andwherein another portion of the linker is operably linked to a secondmolecule.

The term “pegylated” refers to conjugation with polyethylene glycol(PEG), which has been widely used as a drug carrier, given its highdegree of biocompatibility and ease of modification. PEG can be coupled(e.g., covalently linked) to active agents through the hydroxy groups atthe end of the PEG chain via chemical methods; however, PEG itself islimited to at most two active agents per molecule. In a differentapproach, copolymer of PEG and amino acids have been explored as novelbiomaterial which would retain the biocompatibility of PEG, but whichwould have the added advantage of numerous attachment points permolecule (thus providing greater drug loading), and which can besynthetically designed to suit a variety of applications.

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor thereof. The polypeptide can be encoded by a full lengthcoding sequence or by any portion of the coding sequence so as thedesired enzymatic activity is retained.

The term “native” refers to the typical form of a gene, a gene product,or a characteristic of that gene or gene product when isolated from anaturally occurring source. A native form is that which is mostfrequently observed in a nature population and is thus arbitrarilydesignated the normal or wild-type form. In contrast, the term“modified,” “variant,” or “mutant” refers to a gene or gene productwhich displays modification in sequence and functional properties (i.e.,altered characteristics) when compared to the native gene or geneproduct.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques (see, for example, Maniatis et al., 1988 and Ausubel et al.,1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic constructcomprising a nucleic acid coding for a RNA capable of being transcribed.In some cases, RNA molecules are then translated into a protein,polypeptide, or peptide. In other cases, these sequences are nottranslated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host cell. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

The term “therapeutically effective” as used herein refers to an amountof cells and/or therapeutic composition (such as a therapeuticpolynucleotide and/or therapeutic polypeptide) that is employed inmethods to achieve a therapeutic effect, such as wherein at least onesymptom of a condition being treated is at least ameliorated, and/or tothe analysis of the processes or materials used in conjunction withthese cells.

The term “K_(M)” as used herein refers to the Michaelis-Menten constantfor an enzyme and is defined as the concentration of the specificsubstrate at which a given enzyme yields one-half its maximum velocityin an enzyme catalyzed reaction. The term “k_(cat)” as used hereinrefers to the turnover number or the number of substrate molecule eachenzyme site converts to product per unit time, and in which the enzymeis working at maximum efficiency. The term “k_(cat)/K_(M)” as usedherein is the specificity constant which is a measure of how efficientlyan enzyme converts a substrate into product.

The term “cystathionine-γ-lyase” (CGL or cystathionase) refers to anyenzyme that catalyzes the hydrolysis of cystathionine to cysteine. Forexample, it include primate forms of cystathionine-γ-lyase, orparticularly, human forms of cystathionine-γ-lyase.

I. Methionine-γ-Lyase and Cystathionine-γ-Lyase

A lyase is an enzyme that catalyzes the breaking of various chemicalbonds, often forming a new double bond or a new ring structure. Forexample, an enzyme that catalyzed this reaction would be a lyase:ATP→cAMP+PP_(i). Lyases differ from other enzymes in that they onlyrequire one substrate for the reaction in one direction, but twosubstrates for the reverse reaction.

A number of pyrioxal-5′-phosphate (PLP)-dependent enzymes are involvedin the metabolism of cysteine, homocysteine, and methionine, and theseenzymes form an evolutionary related family, designated as Cys/Metmetabolism PLP-dependent enzymes. These enzymes are proteins of about400 amino acids and the PLP group is attached to a lysine residuelocated in the central location of the polypeptide. Members of thisfamily include cystathionine-γ-lyase (CGL), cystathionine-γ-synthase(CGS), cystathionine-β-lyase (CBL), methionine-γ-lyase (MGL),O-acetylhomoserine (OAH)/O-acetyl-serine (OAS) sulfhydrylase (OSHS).Common to all of them is the formation of a Michaelis complex leading toan external substrate aldimine. The further course of the reaction isdetermined by the substrate specificity of the particular enzyme.

For example, the inventors introduced specific mutations into aPLP-dependent lyase family member such as the humancystathionine-γ-lyase to change its substrate specificity. In thismanner the inventors produced novel variants with the de novo ability todegrade L-Met as a substrate. In other embodiments, a modification ofother PLP-dependent enzymes for producing novel methionine degradingactivity may also be contemplated.

As a PLP-dependent enzyme, a methionine gamma-lyase (EC 4.4.1.11) is anenzyme that catalyzes the chemical reaction:L-methionine+H₂O⇄methanethiol+NH3+2-oxobutanoate. Thus, the twosubstrates of this enzyme are L-methionine and H₂O, whereas its 3products are methanethiol, NH3, and 2-oxobutanoate. This enzyme belongsto the family of lyases, specifically the class of carbon-sulfur lyases.The systematic name of this enzyme class is L-methioninemethanethiol-lyase (deaminating 2-oxobutanoate-forming). Other names incommon use include L-methioninase, methionine lyase, methioninase,methionine dethiomethylase, L-methionine gamma-lyase, and L-methioninemethanethiol-lyase (deaminating). This enzyme participates inselenoamino acid metabolism. It employs one cofactor,pyridoxal-5′-phosphate.

Methioninase usually consists of 389-441 amino acids and forms ahomotetramer. Methioninase enzymes are generally composed of fouridentical subunits of molecular weight of ˜45 kDa (Sridhar et al., 2000;Nakamura et al., 1984). The structure of the enzyme was elucidated bycrystallization (Kudou et al., 2007). Each segment of the tetramer iscomposed of three regions: an extended N-terminal domain (residues 1-63)that includes two helices and three beta-strands, a large PLP bindingdomain (residues 64-262) which is made up of a mostly parallel sevenstranded beta-sheet that is sandwiched between eight alpha-helices, anda C-terminal domain (residues 263-398). The cofactor PLP is required forcatalytic function. Amino acids important for catalysis have beenidentified based on the structure. Tyr59 and Arg61 of neighboringsubunits, which are also strongly conserved in other c-family enzymes,contact the phosphate group of PLP. These residues are important as themain anchor within the active site. Lys240, Asp241 and Arg61 of onemonomer and Tyr114 and Cys116 of an adjacent monomer form ahydrogen-bond network in the methioninase active site that confersspecificity to the enzyme.

Cystathionine gamma-lyase (CGL or cystathionase) is an enzyme whichbreaks down cystathionine into cysteine and α-ketobutyrate. Pyridoxalphosphate is a prosthetic group of this enzyme. Although mammals do nothave a methioninase (MGL), they do have cystathionase with sequence,structural, and chemical homology to the bacterial MGL enzymes. As shownin Examples, protein engineering was used to convert cystathionase whichhas no activity for the degradation of L-Methione into an enzyme thatcan degrade this amino acid at a high rate.

II. Methioninase Engineering

Since humans do not produce methione-γ-lyase (MGL or methioninase) it isnecessary to engineer methioninases for human therapy that have highactivity and specificity for degrading methionine under physiologicalconditions, as well as high stability in physiological fluids such asserum and are also non-immunogenic because they are native proteinswhich normally elicit immunological tolerance.

Due to the undesired immunogenicity effects seen in the animal studieswith pMGL, it is desirable to engineer L-methionine degradation activityin a human enzyme. Immunological tolerance to human proteins makes itlikely that such an enzyme will be non-immunogenic or minimallyimmunogenic and therefore well tolerated.

Certain aspects of novel enzymes with MGL activity as engineeredmethioninase address these needs. Although mammals do not have a MGL,they do have a cystathionine-gamma-lyase (CGL) that has sequence,structural, and chemical homology to the bacterial MGL enzymes. CGL is atetramer that catalyzes the last step in the mammalian transsulfurationpathway (Rao et al., 1990). CGL catalyzes the conversion ofL-cystathionine to L-cysteine, alpha-ketobutyrate, and ammonia (FIG. 1).The human CGL (hCGL) cDNA had previously been cloned and expressed, butwith relatively low yields (˜5 mg/L culture) (Lu et al., 1992; Steegbornet al., 1999).

For example, there have been provided methods and compositions relatedto a primate (particularly human) cystathionine-γ-lyase (CGL orcystathionase) modified via mutagenesis to hydrolyze methionine withhigh efficiency, while the cystathionine-γ-lyase does not exhibitmethioninase activity in its native form.

Some embodiments concern modified proteins and polypeptides. Particularembodiments concern a modified protein or polypeptide that exhibits atleast one functional activity that is comparable to the unmodifiedversion, preferably, the methioninase enzyme activity. In furtheraspects, the protein or polypeptide may be further modified to increaseserum stability. Thus, when the present application refers to thefunction or activity of “modified protein” or a “modified polypeptide,”one of ordinary skill in the art would understand that this includes,for example, a protein or polypeptide that possesses an additionaladvantage over the unmodified protein or polypeptide, such as themethioninase enzyme activity. In certain embodiments, the unmodifiedprotein or polypeptide is a native cystathionine-γ-lyase, specifically ahuman cystathionine-γ-lyase. It is specifically contemplated thatembodiments concerning a “modified protein” may be implemented withrespect to a “modified polypeptide,” and vice versa.

Determination of activity may be achieved using assays familiar to thoseof skill in the art, particularly with respect to the protein'sactivity, and may include for comparison purposes, for example, the useof native and/or recombinant versions of either the modified orunmodified protein or polypeptide. For example, the methioninaseactivity may be determined by any assay to detect the production of anysubstrates resulting from conversion of methionine, such asalpha-ketobutyrate, methanethiol, and/or ammonia.

In certain embodiments, a modified polypeptide, such as a modifiedcystathionine-γ-lyase, may be identified based on its increase inmethionine degrading activity. For example, substrate recognition sitesof the unmodified polypeptide may be identified. This identification maybe based on structural analysis or homology analysis. A population ofmutants involving modifications of such substrate recognitions sites maybe generated. In a further embodiment, mutants with increased methioninedegrading activity may be selected from the mutant population. Selectionof desired mutants may include methods such as detection of byproductsor products from methionine degradation.

In a particular embodiment, there may be provided a method using anengineered strain of E. coli comprising chromosomal deletions of genesilvA and metA such that an auxotrophic requirement of L-isoleucine andL-methionine exists. When media supplemented with L-methionine isprovided in the presence of a plasmid encoding a methionine gamma lyaseor an engineered cystathionine gamma-lyase having methionine degradingactivity (but not native cystathionine gamma lyase), it may allow rescueof the L-isoleucine auxotrophy through production of α-ketobutyrate. Themethod may facilitate the identification of mutants with methioninedegrading activity by minimizing the effect from other pathways thatcould also produce α-ketobutyrate.

Modified proteins may possess deletions and/or substitutions of aminoacids; thus, a protein with a deletion, a protein with a substitution,and a protein with a deletion and a substitution are modified proteins.In some embodiments these modified proteins may further includeinsertions or added amino acids, such as with fusion proteins orproteins with linkers, for example. A “modified deleted protein” lacksone or more residues of the native protein, but may possess thespecificity and/or activity of the native protein. A “modified deletedprotein” may also have reduced immunogenicity or antigenicity. Anexample of a modified deleted protein is one that has an amino acidresidue deleted from at least one antigenic region that is, a region ofthe protein determined to be antigenic in a particular organism, such asthe type of organism that may be administered the modified protein.

Substitution or replacement variants typically contain the exchange ofone amino acid for another at one or more sites within the protein andmay be designed to modulate one or more properties of the polypeptide,particularly its effector functions and/or bioavailability.Substitutions may or may not be conservative, that is, one amino acid isreplaced with one of similar shape and charge. Conservativesubstitutions are well known in the art and include, for example, thechanges of: alanine to serine; arginine to lysine; asparagine toglutamine or histidine; aspartate to glutamate; cysteine to serine;glutamine to asparagine; glutamate to aspartate; glycine to proline;histidine to asparagine or glutamine; isoleucine to leucine or valine;leucine to valine or isoleucine; lysine to arginine; methionine toleucine or isoleucine; phenylalanine to tyrosine, leucine or methionine;serine to threonine; threonine to serine; tryptophan to tyrosine;tyrosine to tryptophan or phenylalanine; and valine to isoleucine orleucine.

In addition to a deletion or substitution, a modified protein maypossess an insertion of residues, which typically involves the additionof at least one residue in the polypeptide. This may include theinsertion of a targeting peptide or polypeptide or simply a singleresidue. Terminal additions, called fusion proteins, are discussedbelow.

The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein. Accordingly, sequences thathave between about 70% and about 80%, or between about 81% and about90%, or even between about 91% and about 99% of amino acids that areidentical or functionally equivalent to the amino acids of a controlpolypeptide are included, provided the biological activity of theprotein is maintained. A modified protein may be biologicallyfunctionally equivalent to its native counterpart in certain aspects.

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

III. Enzymatic L-Methionine Depletion for Therapy

In certain aspects, the polypeptides may be used for the treatment ofdiseases including cancers that are sensitive to methionine depletionsuch as hepatocellular carcinoma, melanoma, and renal cell carcinoma,with novel enzymes that deplete L-Methionine. The invention specificallydiscloses treatment methods using modified cystathionine-γ-lyase withmethionine degrading activity. As described below, as currentlyavailable methionine-γ-lyases are typically bacterially-derivedproteins, there remain several problems for their use in human therapy.Certain embodiments of the present invention provide novel enzymes withmethionine-γ-lyase activity for increased therapeutic efficacy.

Methionine (L-Met) depletion has long been studied as a potentialtreatment for cancer. While L-Met is an essential amino acid, manymalignant human cell lines and tumors have been shown to have arelatively greater requirement for methionine (Halpern et al., 1974;Kreis and Goodenow, 1978; Breillout et al., 1990; Kreis et al., 1980;Kreis, 1979). Methionine-dependent tumor cell lines present no or lowlevels of methionine synthase, the enzyme that normally recycleshomocysteine back to L-Met (Halpern et al., 1974; Ashe et al., 1974).Most normal cells can grow on precursors like homocysteine andhomocystine, whereas many malignant cells must scavenge L-Met directlyfrom their extracellular environment. Also, any rapidly growing neoplasmcan be adversely affected by the lack of essential building blocksnecessary for growth. Methionine is particularly important as itsdepletion leads not only to diminished protein synthesis, but alsodysregulates S-adenosylmethionine (SAM) dependent methylation pathways,which are particularly important for gene regulation.

The differences in methionine requirement between normal and cancercells provide a therapeutic opportunity. Enzymatic methionine depletionhas been explored in a number of animal model studies as well as phase Iclinical trials (Tan et al., 1997a; Tan et al., 1996a; Lishko et al.,1993; Tan et al., 1996b; Yoshioka et al., 1998; Yang et al., 2004a; Yanget al., 2004b; Tan et al., 1997b).

Because humans lack a methionine hydrolyzing enzyme, bacterialL-methionine-gamma-lyases, MGL, from various sources have been evaluatedfor cancer therapy. Methionine-gamma-lyase catalyzes the conversion ofmethionine to methanethiol, alpha-ketobutyrate, and ammonia. Bacterialenzymes from various sources have been purified and tested as methioninedepleting agents against cancer cell lines. The P. putida (pMGL) sourcewas selected for therapeutic applications due to its high catalyticactivity, low K_(M) and a relatively high k_(cat) value (Esaki and Soda,1987; Ito et al., 1976), in comparison to other sources. Furthermore,the gene for pMGL has been cloned into E. coli and the protein wasexpressed at a high protein yield (Tan et al., 1997a; Hori et al.,1996).

In vivo studies have been performed on animal models, as well as humans.Tan et al. performed studies with human tumors xenografted in nude miceand found that lung, colon, kidney, brain, prostate, and melanomacancers were all sensitive to pMGL (Tan et al., 1997a). Additionally, notoxicity was detected at effective doses (as was determined by anabsence of weight loss in the animals. Half-life in these experimentswas determined to be only 2 hr as measured from collected blood samples.Additionally infusion of PLP is required in order to maintain MGLactivity. In spite of the very short half-life, Tan et al. reportedinhibition of tumor growth in comparison to a saline control.

Yang et al. studied the pharmacokinetics, the pharmacodymanics in termsof methionine depletion, the antigenicity, and toxicity of MGL in aprimate model (Yang et al., 2004b). Dose-ranging studies were performedat 1000-4000 units/kg administered intravenously. The highest dose wasable to reduce plasma methionine to an undetectable level (less than 0.5μM) by 30 minutes after injection, with the methionine level remainingundetectable for 8 hr. Pharmacokinetic analysis showed that pMGL waseliminated with a half-life of 2.5 hr. An administration of that doseevery 8 hr/day for 2 weeks resulted in a steady-state depletion ofplasma methionine to less than 2 μM. Mild toxicity was observed throughdecreased food intake and slight weight loss. Unfortunately,re-challenge on day 28 resulted in anaphylactic shock and death in oneanimal indicating that pMGL is highly immunogenic, which is asignificant disadvantage for human therapy. Subsequent pretreatment withhydrocortisone prevented the anaphylactic reaction, although vomitingwas frequently observed. Additional re-challenges were carried out atdays 66, 86, and 116. Anti-rMGL antibodies were detected after the firstchallenge, and increased in concentration for the duration of treatment.

In response to these observed obstacles to therapeutic implementation ofMGL, Yang et al. studied the PEGylation of the enzyme and its effect onhalf-life and immunogenicity. The enzyme was coupled tomethoxypolyethylene glycol succinimidyl glutarate (MEGC-PEG-5000). Doseranging studies were again performed and 4,000 units/kg (90 mg/kg) wassufficient to reduce plasma methionine to <5 μmol/L for 12 hours.Pharmacokinetic analysis showed a 36 fold improvement in the serumclearance half-life of the PEGylated enzyme, as compared to theunpegylated. Pegylating also attenuated immunogenicity somewhat as onlyslight toxicities of decreased food intake and minor weight loss wereobserved. However the activity half-life was not improved as L-Metlevels were only kept below detection levels for 12 hrs as opposed to 8hrs for the unpegylated enzyme. These results, though promising in theability of a L-Met depleting enzyme as an anti-neoplastic agent, arechallenged by significant shortcomings of immunogenicity andpharmacokinetics.

Certain aspects of the present invention provide a modifiedcystathionine-γ-lyase with methionine degrading activity for treatingdiseases, such as tumor. Particularly, the modified polypeptide may havehuman polypeptide sequences and thus may prevent allergic reactions inhuman patients, allow repeated dosing, and increase the therapeuticefficacy.

Tumors for which the present treatment methods are useful include anymalignant cell type such as those found in a solid tumor or ahematological tumor. Exemplary solid tumors can include, but are notlimited to a tumor of an organ selected from the group consisting ofpancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney,larynx, sarcoma, lung, bladder, melanoma, prostate and breast. Exemplaryhematological tumors include tumors of the bone marrow, T or B cellmalignancies, leukemia, lymphomas, blastomas, myelomas, and the like.

The engineered human methioninase derived from cystathionase may be usedherein as an antitumor agent in a variety of modalities for depletingmethionine from a tumor cell, tumor tissue or the circulation of amammal with cancer, or for depletion of methionine where its depletionis considered desirable.

Depletion can be conducted in vivo, in the circulation of a mammal, invitro in cases where methionine depletion in tissue culture or otherbiological mediums is desired, and in ex vivo procedures wherebiological fluids, cells or tissues are manipulated outside the body andsubsequently returned to the body of the patient mammal. Depletion ofmethionine from circulation, culture media, biological fluids or cellsis conducted to reduce the amount of methionine accessible to thematerial being treated, and therefore comprises contacting the materialto be depleted with a methionine-depleting amount of the engineeredhuman methioninase under methionine-depleting conditions as to degradethe ambient methionine in the material being contacted.

Because tumor cells are dependent upon their nutrient medium formethionine, the depletion may be directed to the nutrient source for thecells, and not necessarily the cells themselves. Therefore, in an invivo application, treating a tumor cell includes contacting the nutrientmedium for a population of tumor cells with the engineered methioninase.In this embodiment, the medium can be blood, lymphatic fluid, spinalfluid and the like bodily fluid where methionine depletion is desired.

A methionine-depleting efficiency can vary widely depending upon theapplication, and typically depends upon the amount of methionine presentin the material, the desired rate of depletion, and the tolerance of thematerial for exposure to methioninase. Methionine levels in a material,and therefore rates of methionine depletion from the material, canreadily be monitored by a variety of chemical and biochemical methodswell known in the art. Exemplary methionine-depleting amounts aredescribed further herein, and can range from 0.001 to 100 units (U) ofengineered methioninase, preferably about 0.01 to 10 U, and morepreferably about 0.1 to 5 U engineered methioninase per milliliter (ml)of material to be treated.

Methionine-depleting conditions are buffer and temperature conditionscompatible with the biological activity of a methioninase enzyme, andinclude moderate temperature, salt and pH conditions compatible with theenzyme, for example, physiological conditions. Exemplary conditionsinclude about 4-40° C., ionic strength equivalent to about 0.05 to 0.2 MNaCl, and a pH of about 5 to 9, while physiological conditions areincluded.

In a particular embodiment, the invention contemplates methods of usingengineered methioninase as an antitumor agent, and therefore comprisescontacting a population of tumor cells with a therapeutically effectiveamount of engineered methioninase for a time period sufficient toinhibit tumor cell growth.

In one embodiment, the contacting in vivo is accomplished byadministering, by intravenous or intraperitoneal injection, atherapeutically effective amount of a physiologically tolerablecomposition containing engineered methioninase of this invention to apatient, thereby depleting the circulating methionine source of thetumor cells present in the patient. The contacting of engineeredmethioninase can also be accomplished by administering the engineeredmethioninase into the tissue containing the tumor cells.

A therapeutically effective amount of an engineered methioninase is apredetermined amount calculated to achieve the desired effect, i.e., todeplete methionine in the tumor tissue or in a patient's circulation,and thereby cause the tumor cells to stop dividing. Thus, the dosageranges for the administration of engineered methioninase of theinvention are those large enough to produce the desired effect in whichthe symptoms of tumor cell division and cell cycling are reduced. Thedosage should not be so large as to cause adverse side effects, such ashyperviscosity syndromes, pulmonary edema, congestive heart failure, andthe like. Generally, the dosage will vary with the age, condition, sexand extent of the disease in the patient and can be determined by one ofskill in the art. The dosage can be adjusted by the individual physicianin the event of any complication.

For example, a therapeutically effective amount of an engineeredmethioninase may be an amount such that when administered in aphysiologically tolerable composition is sufficient to achieve aintravascular (plasma) or local concentration of from about 0.001 toabout 100 units (U) per ml, preferably above about 0.1 U, and morepreferably above 1 U engineered methioninase per ml. Typical dosages canbe administered based on body weight, and are in the range of about5-1000 U/kilogram (kg)/day, preferably about 5-100 U/kg/day, morepreferably about 10-50 U/kg/day, and more preferably about 20-40U/kg/day.

The engineered methioninase can be administered parenterally byinjection or by gradual infusion over time. The engineered methioninasecan be administered intravenously, intraperitoneally, orally,intramuscularly, subcutaneously, intracavity, transdermally, dermally,can be delivered by peristaltic means, or can be injected directly intothe tissue containing the tumor cells or can be administered by a pumpconnected to a catheter that may contain a potential biosensor ormethionine.

The therapeutic compositions containing engineered methioninase areconventionally administered intravenously, as by injection of a unitdose, for example. The term “unit dose” when used in reference to atherapeutic composition refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,carrier, or vehicle.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's system to utilize the active ingredient, and degree oftherapeutic effect desired. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitionerand are peculiar to each individual. However, suitable dosage ranges forsystemic application are disclosed herein and depend on the route ofadministration. Suitable regimes for initial administration and boostershots are also contemplated and are typified by an initialadministration followed by repeated doses at one or more hour intervalsby a subsequent injection or other administration. Exemplary multipleadministrations are described herein and are particularly preferred tomaintain continuously high serum and tissue levels of engineeredmethioninase and conversely low serum and tissue levels of methionine.Alternatively, continuous intravenous infusion sufficient to maintainconcentrations in the blood in the ranges specified for in vivotherapies are contemplated.

IV. Conjugates

Compositions and methods of the present invention involve furthermodification of the engineered methioninase for improvement, such as byforming conjugates with heterologous peptide segments or polymers suchas polyethylene glycol. In further aspects, the engineered methioninasemay be linked to PEG to increase the hydrodynamic radius of the enzymeand hence increase the serum persistence. In certain aspects, thedisclosed polypeptide may be conjugated to any targeting agent such as aligand having the ability to specifically and stably bind to an externalreceptor or binding site on a tumor cell (U.S. Patent Publ.2009/0304666)

A. Fusion Proteins

Certain embodiments of the present invention concern fusion proteins.These molecules may have the modified cystathionase linked at the N- orC-terminus to a heterologous domain. For example, fusions may alsoemploy leader sequences from other species to permit the recombinantexpression of a protein in a heterologous host. Another useful fusionincludes the addition of a protein affinity tag like six histidineresidues or an immunologically active domain, such as an antibodyepitope, preferably cleavable, to facilitate purification of the fusionprotein. Non-limiting affinity tags include polyhistidine, chitinbinding protein (CBP), maltose binding protein (MBP), andglutathione-S-transferase (GST).

In a particular embodiment, the modified cystathionine gamma-lyase maybe linked to a peptide that increases the in vivo half-life, such as anXTEN polypeptide (Schellenberger et al., 2009).

Methods of generating fusion proteins are well known to those of skillin the art. Such proteins can be produced, for example, by de novosynthesis of the complete fusion protein, or by attachment of the DNAsequence encoding the heterologous domain, followed by expression of theintact fusion protein.

Production of fusion proteins that recover the functional activities ofthe parent proteins may be facilitated by connecting genes with abridging DNA segment encoding a peptide linker that is spliced betweenthe polypeptides connected in tandem. The linker would be of sufficientlength to allow proper folding of the resulting fusion protein.

B. Linkers

In certain embodiments, the engineered methioninase may be chemicallyconjugated using bifunctional cross-linking reagents or fused at theprotein level with peptide linkers.

Bifunctional cross-linking reagents have been extensively used for avariety of purposes including preparation of affinity matrices,modification and stabilization of diverse structures, identification ofligand and receptor binding sites, and structural studies. Suitablepeptide linkers may also be used to link the engineered methioninase,such as Gly-Ser linkers.

Homobifunctional reagents that carry two identical functional groupsproved to be highly efficient in inducing cross-linking betweenidentical and different macromolecules or subunits of a macromolecule,and linking of polypeptide ligands to their specific binding sites.Heterobifunctional reagents contain two different functional groups. Bytaking advantage of the differential reactivities of the two differentfunctional groups, cross-linking can be controlled both selectively andsequentially. The bifunctional cross-linking reagents can be dividedaccording to the specificity of their functional groups, e.g., amino,sulfhydryl, guanidino, indole, carboxyl specific groups. Of these,reagents directed to free amino groups have become especially popularbecause of their commercial availability, ease of synthesis and the mildreaction conditions under which they can be applied.

A majority of heterobifunctional cross-linking reagents contains aprimary amine-reactive group and a thiol-reactive group. In anotherexample, heterobifunctional cross-linking reagents and methods of usingthe cross-linking reagents are described (U.S. Pat. No. 5,889,155,specifically incorporated herein by reference in its entirety). Thecross-linking reagents combine a nucleophilic hydrazide residue with anelectrophilic maleimide residue, allowing coupling in one example, ofaldehydes to free thiols. The cross-linking reagent can be modified tocross-link various functional groups.

Additionally, any other linking/coupling agents and/or mechanisms knownto those of skill in the art may be used to combine human engineeredmethioninase, such as, for example, antibody-antigen interaction, avidinbiotin linkages, amide linkages, ester linkages, thioester linkages,ether linkages, thioether linkages, phosphoester linkages, phosphoramidelinkages, anhydride linkages, disulfide linkages, ionic and hydrophobicinteractions, bispecific antibodies and antibody fragments, orcombinations thereof.

It is preferred that a cross-linker having reasonable stability in bloodwill be employed. Numerous types of disulfide-bond containing linkersare known that can be successfully employed to conjugate targeting andtherapeutic/preventative agents. Linkers that contain a disulfide bondthat is sterically hindered may prove to give greater stability in vivo.These linkers are thus one group of linking agents.

In addition to hindered cross-linkers, non-hindered linkers also can beemployed in accordance herewith. Other useful cross-linkers, notconsidered to contain or generate a protected disulfide, include SATA,SPDP and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of suchcross-linkers is well understood in the art. Another embodiment involvesthe use of flexible linkers.

Once chemically conjugated, the peptide generally will be purified toseparate the conjugate from unconjugated agents and from othercontaminants. A large number of purification techniques are availablefor use in providing conjugates of a sufficient degree of purity torender them clinically useful.

Purification methods based upon size separation, such as gel filtration,gel permeation or high performance liquid chromatography, will generallybe of most use. Other chromatographic techniques, such as Blue-Sepharoseseparation, may also be used. Conventional methods to purify the fusionproteins from inclusion bodies may be useful, such as using weakdetergents like sodium N-lauroyl-sarcosine (SLS).

C. Pegylation

In certain aspects of the invention, methods and compositions related topegylation of engineered methioninase are disclosed. For example, theengineered methioninase may be pegylated in accordance with the methodsdisclosed herein.

Pegylation is the process of covalent attachment of poly(ethyleneglycol) polymer chains to another molecule, normally a drug ortherapeutic protein. Pegylation is routinely achieved by incubation of areactive derivative of PEG with the target macromolecule. The covalentattachment of PEG to a drug or therapeutic protein can “mask” the agentfrom the host's immune system (reduced immunogenicity and antigenicity),increase the hydrodynamic size (size in solution) of the agent whichprolongs its circulatory time by reducing renal clearance. Pegylationcan also provide water solubility to hydrophobic drugs and proteins.

The first step of the pegylation is the suitable functionalization ofthe PEG polymer at one or both terminals. PEGs that are activated ateach terminus with the same reactive moiety are known as“homobifunctional”, whereas if the functional groups present aredifferent, then the PEG derivative is referred as “heterobifunctional”or “heterofunctional.” The chemically active or activated derivatives ofthe PEG polymer are prepared to attach the PEG to the desired molecule.

The choice of the suitable functional group for the PEG derivative isbased on the type of available reactive group on the molecule that willbe coupled to the PEG. For proteins, typical reactive amino acidsinclude lysine, cysteine, histidine, arginine, aspartic acid, glutamicacid, serine, threonine, tyrosine. The N-terminal amino group and theC-terminal carboxylic acid can also be used.

The techniques used to form first generation PEG derivatives aregenerally reacting the PEG polymer with a group that is reactive withhydroxyl groups, typically anhydrides, acid chlorides, chloroformatesand carbonates. In the second generation pegylation chemistry moreefficient functional groups such as aldehyde, esters, amides etc madeavailable for conjugation.

As applications of pegylation have become more and more advanced andsophisticated, there has been an increase in need for heterobifunctionalPEGs for conjugation. These heterobifunctional PEGs are very useful inlinking two entities, where a hydrophilic, flexible and biocompatiblespacer is needed. Preferred end groups for heterobifunctional PEGs aremaleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acidsand NHS esters.

The most common modification agents, or linkers, are based on methoxyPEG (mPEG) molecules. Their activity depends on adding aprotein-modifying group to the alcohol end. In some instancespolyethylene glycol (PEG diol) is used as the precursor molecule. Thediol is subsequently modified at both ends in order to make a hetero- orhomo-dimeric PEG-linked molecule (as shown in the example with PEGbis-vinylsulfone).

Proteins are generally PEGylated at nucleophilic sites such asunprotonated thiols (cysteinyl residues) or amino groups. Examples ofcysteinyl-specific modification reagents include PEG maleimide, PEGiodoacetate, PEG thiols, and PEG vinylsulfone. All four are stronglycysteinyl-specific under mild conditions and neutral to slightlyalkaline pH but each has some drawbacks. The amide formed with themaleimides can be somewhat unstable under alkaline conditions so theremay be some limitation to formulation options with this linker. Theamide linkage formed with iodo PEGs is more stable, but free iodine canmodify tyrosine residues under some conditions. PEG thiols formdisulfide bonds with protein thiols, but this linkage can also beunstable under alkaline conditions. PEG-vinylsulfone reactivity isrelatively slow compared to maleimide and iodo PEG; however, thethioether linkage formed is quite stable. Its slower reaction rate alsocan make the PEG-vinylsulfone reaction easier to control.

Site-specific pegylation at native cysteinyl residues is seldom carriedout, since these residues are usually in the form of disulfide bonds orare required for biological activity. On the other hand, site-directedmutagenesis can be used to incorporate cysteinyl pegylation sites forthiol-specific linkers. The cysteine mutation must be designed such thatit is accessible to the pegylation reagent and is still biologicallyactive after pegylation.

Amine-specific modification agents include PEG NHS ester, PEG tresylate,PEG aldehyde, PEG isothiocyanate, and several others. All react undermild conditions and are very specific for amino groups. The PEG NHSester is probably one of the more reactive agents; however, its highreactivity can make the pegylation reaction difficult to control atlarge scale. PEG aldehyde forms an imine with the amino group, which isthen reduced to a secondary amine with sodium cyanoborohydride. Unlikesodium borohydride, sodium cyanoborohydride will not reduce disulfidebonds. However, this chemical is highly toxic and must be handledcautiously, particularly at lower pH where it becomes volatile.

Due to the multiple lysine residues on most proteins, site-specificpegylation can be a challenge. Fortunately, because these reagents reactwith unprotonated amino groups, it is possible to direct the pegylationto lower-pK amino groups by performing the reaction at a lower pH.Generally the pK of the alpha-amino group is 1-2 pH units lower than theepsilon-amino group of lysine residues. By pegylating the molecule at pH7 or below, high selectivity for the N-terminus frequently can beattained. However, this is only feasible if the N-terminal portion ofthe protein is not required for biological activity. Still, thepharmacokinetic benefits from pegylation frequently outweigh asignificant loss of in vitro bioactivity, resulting in a product withmuch greater in vivo bioactivity regardless of pegylation chemistry.

There are several parameters to consider when developing a pegylationprocedure. Fortunately, there are usually no more than four or five keyparameters. The “design of experiments” approach to optimization ofpegylation conditions can be very useful. For thiol-specific pegylationreactions, parameters to consider include: protein concentration,PEG-to-protein ratio (on a molar basis), temperature, pH, reaction time,and in some instances, the exclusion of oxygen. (Oxygen can contributeto intermolecular disulfide formation by the protein, which will reducethe yield of the PEGylated product.) The same factors should beconsidered (with the exception of oxygen) for amine-specificmodification except that pH may be even more critical, particularly whentargeting the N-terminal amino group.

For both amine- and thiol-specific modifications, the reactionconditions may affect the stability of the protein. This may limit thetemperature, protein concentration, and pH. In addition, the reactivityof the PEG linker should be known before starting the pegylationreaction. For example, if the pegylation agent is only 70 percentactive, the amount of PEG used should ensure that only active PEGmolecules are counted in the protein-to-PEG reaction stoichiometry.

V. Proteins and Peptides

In certain embodiments, the present invention concerns novelcompositions comprising at least one protein or peptide, such asengineered methioninase. These peptides may be comprised in a fusionprotein or conjugated to an agent as described supra.

As used herein, a protein or peptide generally refers, but is notlimited to, a protein of greater than about 200 amino acids, up to afull length sequence translated from a gene; a polypeptide of greaterthan about 100 amino acids; and/or a peptide of from about 3 to about100 amino acids. For convenience, the terms “protein,” “polypeptide” and“peptide are used interchangeably herein.

As used herein, an “amino acid residue” refers to any naturallyoccurring amino acid, any amino acid derivative or any amino acid mimicknown in the art. In certain embodiments, the residues of the protein orpeptide are sequential, without any non-amino acid interrupting thesequence of amino acid residues. In other embodiments, the sequence maycomprise one or more non-amino acid moieties. In particular embodiments,the sequence of residues of the protein or peptide may be interrupted byone or more non-amino acid moieties.

Accordingly, the term “protein or peptide” encompasses amino acidsequences comprising at least one of the 20 common amino acids found innaturally occurring proteins, or at least one modified or unusual aminoacid, including but not limited to those shown on Table 1 below.

Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, or the chemical synthesisof proteins or peptides. The nucleotide and protein, polypeptide andpeptide sequences corresponding to various genes have been previouslydisclosed, and may be found at computerized databases known to those ofordinary skill in the art. One such database is the National Center forBiotechnology Information's Genbank and GenPept databases (available onthe world wide web at ncbi.nlm.nih.gov/). The coding regions for knowngenes may be amplified and/or expressed using the techniques disclosedherein or as would be known to those of ordinary skill in the art.Alternatively, various commercial preparations of proteins, polypeptidesand peptides are known to those of skill in the art.

VI. Nucleic Acids and Vectors

In certain aspects of the invention, nucleic acid sequences encoding aan engineered methioninase or a fusion protein containing a modifiedcystathionase may be disclosed. Depending on which expression system tobe used, nucleic acid sequences can be selected based on conventionalmethods. For example, the engineered methioninase is derived from humancystathionase and contains multiple codons that are rarely utilized inE. coli that may interfere with expression, therefore the respectivegenes or variants thereof may be codon optimized for E. coli expression.Various vectors may be also used to express the protein of interest,such as engineered methioninase. Exemplary vectors include, but are notlimited, plasmid vectors, viral vectors, transposon or liposome-basedvectors.

VII. Host Cells

Host cells may be any that may be transformed to allow the expressionand secretion of engineered methioninase and conjugates thereof. Thehost cells may be bacteria, mammalian cells, yeast, or filamentousfungi. Various bacteria include Escherichia and Bacillus. Yeastsbelonging to the genera Saccharomyces, Kiuyveromyces, Hansenula, orPichia would find use as an appropriate host cell. Various species offilamentous fungi may be used as expression hosts including thefollowing genera: Aspergillus, Trichoderma, Neurospora, Penicillium,Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochliobolus andPyricularia.

Examples of usable host organisms include bacteria, e.g., Escherichiacoli MC1061, derivatives of Bacillus subtilis BRB1 (Sibakov et al.,1984), Staphylococcus aureus SAI123 (Lordanescu, 1975) or Streptococcuslividans (Hopwood et al., 1985); yeasts, e.g., Saccharomyces cerevisiaeAH 22 (Mellor et al., 1983) and Schizosaccharomyces pombe; filamentousfungi, e.g., Aspergillus nidulans, Aspergillus awamori (Ward, 1989),Trichoderma reesei (Penttila et al., 1987; Harkki et al, 1989).

Examples of mammalian host cells include Chinese hamster ovary cells(CHO-K1; ATCC CCL61), rat pituitary cells (GH₁; ATCC CCL82), HeLa S3cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCCCRL 1548)SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650) and murineembryonic cells (NIH-3T3; ATCC CRL 1658). The foregoing beingillustrative but not limitative of the many possible host organismsknown in the art. In principle, all hosts capable of secretion can beused whether prokaryotic or eukaryotic.

Mammalian host cells expressing the engineered methioninases and/ortheir fusion proteins are cultured under conditions typically employedto culture the parental cell line. Generally, cells are cultured in astandard medium containing physiological salts and nutrients, such asstandard RPMI, MEM, IMEM or DMEM, typically supplemented with 5-10%serum, such as fetal bovine serum. Culture conditions are also standard,e.g., cultures are incubated at 37° C. in stationary or roller culturesuntil desired levels of the proteins are achieved.

VIII. Protein Purification

Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the homogenization andcrude fractionation of the cells, tissue or organ to polypeptide andnon-polypeptide fractions. The protein or polypeptide of interest may befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity) unless otherwise specified. Analytical methods particularlysuited to the preparation of a pure peptide are ion-exchangechromatography, gel exclusion chromatography, polyacrylamide gelelectrophoresis, affinity chromatography, immunoaffinity chromatographyand isoelectric focusing. A particularly efficient method of purifyingpeptides is fast performance liquid chromatography (FPLC) or even highperformance liquid chromatography (HPLC).

A purified protein or peptide is intended to refer to a composition,isolatable from other components, wherein the protein or peptide ispurified to any degree relative to its naturally-obtainable state. Anisolated or purified protein or peptide, therefore, also refers to aprotein or peptide free from the environment in which it may naturallyoccur. Generally, “purified” will refer to a protein or peptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a composition in which theprotein or peptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, or more of the proteins in the composition.

Various techniques suitable for use in protein purification are wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like, orby heat denaturation, followed by: centrifugation; chromatography stepssuch as ion exchange, gel filtration, reverse phase, hydroxyapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of these and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

Various methods for quantifying the degree of purification of theprotein or peptide are known to those of skill in the art in light ofthe present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity therein,assessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification, andwhether or not the expressed protein or peptide exhibits a detectableactivity.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products may have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

In certain embodiments a protein or peptide may be isolated or purified,for example, an engineered methioninase, a fusion protein containing theengineered methioninase, or an engineered methioninase post pegylation.For example, a His tag or an affinity epitope may be comprised in suchan engineered methioninase to facilitate purification. Affinitychromatography is a chromatographic procedure that relies on thespecific affinity between a substance to be isolated and a molecule towhich it can specifically bind. This is a receptor-ligand type ofinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (e.g., altered pH, ionic strength, temperature, etc.). Thematrix should be a substance that itself does not adsorb molecules toany significant extent and that has a broad range of chemical, physicaland thermal stability. The ligand should be coupled in such a way as tonot affect its binding properties. The ligand should also providerelatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand.

Size exclusion chromatography (SEC) is a chromatographic method in whichmolecules in solution are separated based on their size, or in moretechnical terms, their hydrodynamic volume. It is usually applied tolarge molecules or macromolecular complexes such as proteins andindustrial polymers. Typically, when an aqueous solution is used totransport the sample through the column, the technique is known as gelfiltration chromatography, versus the name gel permeation chromatographywhich is used when an organic solvent is used as a mobile phase.

The underlying principle of SEC is that particles of different sizeswill elute (filter) through a stationary phase at different rates. Thisresults in the separation of a solution of particles based on size.Provided that all the particles are loaded simultaneously or nearsimultaneously, particles of the same size should elute together. Eachsize exclusion column has a range of molecular weights that can beseparated. The exclusion limit defines the molecular weight at the upperend of this range and is where molecules are too large to be trapped inthe stationary phase. The permeation limit defines the molecular weightat the lower end of the range of separation and is where molecules of asmall enough size can penetrate into the pores of the stationary phasecompletely and all molecules below this molecular mass are so small thatthey elute as a single band.

High-performance liquid chromatography (or High pressure liquidchromatography, HPLC) is a form of column chromatography used frequentlyin biochemistry and analytical chemistry to separate, identify, andquantify compounds. HPLC utilizes a column that holds chromatographicpacking material (stationary phase), a pump that moves the mobilephase(s) through the column, and a detector that shows the retentiontimes of the molecules. Retention time varies depending on theinteractions between the stationary phase, the molecules being analyzed,and the solvent(s) used.

IX. Pharmaceutical Compositions

It is contemplated that the novel methioninase can be administeredsystemically or locally to inhibit tumor cell growth and, mostpreferably, to kill cancer cells in cancer patients with locallyadvanced or metastatic cancers. They can be administered intravenously,intrathecally, and/or intraperitoneally. They can be administered aloneor in combination with anti-proliferative drugs. In one embodiment, theyare administered to reduce the cancer load in the patient prior tosurgery or other procedures. Alternatively, they can be administeredafter surgery to ensure that any remaining cancer (e.g. cancer that thesurgery failed to eliminate) does not survive.

It is not intended that the present invention be limited by theparticular nature of the therapeutic preparation. For example, suchcompositions can be provided in formulations together withphysiologically tolerable liquid, gel or solid carriers, diluents, andexcipients. These therapeutic preparations can be administered tomammals for veterinary use, such as with domestic animals, and clinicaluse in humans in a manner similar to other therapeutic agents. Ingeneral, the dosage required for therapeutic efficacy will varyaccording to the type of use and mode of administration, as well as theparticularized requirements of individual subjects.

Such compositions are typically prepared as liquid solutions orsuspensions, as injectables. Suitable diluents and excipients are, forexample, water, saline, dextrose, glycerol, or the like, andcombinations thereof. In addition, if desired the compositions maycontain minor amounts of auxiliary substances such as wetting oremulsifying agents, stabilizing or pH buffering agents.

Where clinical applications are contemplated, it may be necessary toprepare pharmaceutical compositions comprising proteins, antibodies anddrugs in a form appropriate for the intended application. Generally,pharmaceutical compositions may comprise an effective amount of one ormore cystathionase variants or additional agent dissolved or dispersedin a pharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcontains at least one cystathionase variant isolated by the methoddisclosed herein, or additional active ingredient will be known to thoseof skill in the art in light of the present disclosure, as exemplifiedby Remington's Pharmaceutical Sciences, 18^(th) Ed., 1990, incorporatedherein by reference. Moreover, for animal (e.g., human) administration,it will be understood that preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18^(th) Ed., 1990, incorporated herein by reference). Except insofar asany conventional carrier is incompatible with the active ingredient, itsuse in the pharmaceutical compositions is contemplated.

Certain embodiments of the present invention may comprise differenttypes of carriers depending on whether it is to be administered insolid, liquid or aerosol form, and whether it need to be sterile forsuch routes of administration as injection. The compositions can beadministered intravenously, intradermally, transdermally, intrathecally,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, topically, intramuscularly, subcutaneously, mucosally,orally, topically, locally, inhalation (e.g., aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art (see,for example, Remington's Pharmaceutical Sciences, 18^(th) Ed., 1990,incorporated herein by reference).

The modified polypeptides may be formulated into a composition in a freebase, neutral or salt form. Pharmaceutically acceptable salts, includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentary administrations such as drugrelease capsules and the like.

Further in accordance with certain aspects of the present invention, thecomposition suitable for administration may be provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a the composition contained therein, itsuse in administrable composition for use in practicing the methods isappropriate. Examples of carriers or diluents include fats, oils, water,saline solutions, lipids, liposomes, resins, binders, fillers and thelike, or combinations thereof. The composition may also comprise variousantioxidants to retard oxidation of one or more component. Additionally,the prevention of the action of microorganisms can be brought about bypreservatives such as various antibacterial and antifungal agents,including but not limited to parabens (e.g., methylparabens,propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal orcombinations thereof.

In accordance with certain aspects of the present invention, thecomposition is combined with the carrier in any convenient and practicalmanner, i.e., by solution, suspension, emulsification, admixture,encapsulation, absorption and the like. Such procedures are routine forthose skilled in the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include cystathionasevariants, one or more lipids, and an aqueous solvent. As used herein,the term “lipid” will be defined to include any of a broad range ofsubstances that is characteristically insoluble in water and extractablewith an organic solvent. This broad class of compounds are well known tothose of skill in the art, and as the term “lipid” is used herein, it isnot limited to any particular structure. Examples include compoundswhich contain long-chain aliphatic hydrocarbons and their derivatives. Alipid may be naturally occurring or synthetic (i.e., designed orproduced by man). However, a lipid is usually a biological substance.Biological lipids are well known in the art, and include for example,neutral fats, phospholipids, phosphoglycerides, steroids, terpenes,lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids withether and ester-linked fatty acids and polymerizable lipids, andcombinations thereof. Of course, compounds other than those specificallydescribed herein that are understood by one of skill in the art aslipids are also encompassed by the compositions and methods.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the engineered methioninase or a fusion proteinthereof may be dispersed in a solution containing a lipid, dissolvedwith a lipid, emulsified with a lipid, mixed with a lipid, combined witha lipid, covalently bonded to a lipid, contained as a suspension in alipid, contained or complexed with a micelle or liposome, or otherwiseassociated with a lipid or lipid structure by any means known to thoseof ordinary skill in the art. The dispersion may or may not result inthe formation of liposomes.

The actual dosage amount of a composition administered to an animalpatient can be determined by physical and physiological factors such asbody weight, severity of condition, the type of disease being treated,previous or concurrent therapeutic interventions, idiopathy of thepatient and on the route of administration. Depending upon the dosageand the route of administration, the number of administrations of apreferred dosage and/or an effective amount may vary according to theresponse of the subject. The practitioner responsible for administrationwill, in any event, determine the concentration of active ingredient(s)in a composition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

X. Kits

Certain aspects of the present invention may provide kits, such astherapeutic kits. For example, a kit may comprise one or morepharmaceutical composition as described herein and optionallyinstructions for their use. Kits may also comprise one or more devicesfor accomplishing administration of such compositions. For example, asubject kit may comprise a pharmaceutical composition and catheter foraccomplishing direct intravenous injection of the composition into acancerous tumor. In other embodiments, a subject kit may comprisepre-filled ampoules of an engineered methioninase, optionally formulatedas a pharmaceutical, or lyophilized, for use with a delivery device.

Kits may comprise a container with a label. Suitable containers include,for example, bottles, vials, and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer may hold a composition which includes an antibody that iseffective for therapeutic or non-therapeutic applications, such asdescribed above. The label on the container may indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, and may also indicate directions for either in vivo or invitro use, such as those described above. The kit of the invention willtypically comprise the container described above and one or more othercontainers comprising materials desirable from a commercial and userstandpoint, including buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for use.

XI. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Gene Synthesis and Expression of Native HumanCystathionine-γ-Lyase and Modified Human Cystathionine-γ-Lyase

Using sequence and structural alignments of cystathionine-gamma-lyase(CGL) and methionine-γ-lyase (MGL) enzymes as a guide, CGL, particularlyhuman CGL (hCGL) were converted to an enzyme for the efficient degradingof methionine.

The human cystathionase gene contains multiple codons that arerarely-utilized in E. coli and can interfere with expression. Thus, inorder to optimize protein expression in E. coli, the hGCL gene wassynthesized using codon optimized oligonucleotides designed using theDNA-Works software (Hoover et al., 2002). An NcoI 5′ restriction site,an in-frame N-terminal His6 tag and a 3′ EcoRI site were introduced intothe ORF. After cloning into a pET28a vector (Novagen), E. coli (BL21)transformants containing an appropriate cystathionase expression vectorare grown at 37° C. using Terrific Broth (TB) media containing 50 μg/mlkanamycin in shake flasks at 250 rpm until reaching an OD₆₀₀ of 0.5-0.6.At this point the cultures are switched to a shaker at 25° C., inducedwith 0.5 mM IPTG, and allowed to express protein for an additional 12hrs. Cell pellets are then collected by centrifugation and re-suspendedin an IMAC (Immobilized Metal. Affinity Chromatography) buffer (10 mMNaPO₄/10 mM imidazole/300 mM NaCl, pH 8). After lysis by a Frenchpressure cell, lysates were centrifuged at 20,000×g for 20 min at 4° C.,and the resulting supernatant applied to a nickel IMAC column, washedwith 10-20 column volumes of IMAC buffer, and then eluted with an IMACelution buffer (50 mM NaPO₄/250 mM imidazole/300 mM NaCl, pH 8).Fractions containing enzyme are then incubated with 10 mMpyridoxal-5′-phosphate (PLP) for an hour at 25° C. Using a 10,000 MWCOcentrifugal filter device (Amicon), proteins are then buffer exchangedseveral times into a 100 mM PBS, 10% glycerol, pH 7.3 solution. Aliquotsof cystathionase enzyme are then flash frozen in liquid nitrogen andstored at −80° C. Cystathionase purified in this manner is >95%homogeneous as assessed by SDS-PAGE and coomassie staining. The yield iscalculated to be ˜400 mg/L culture based upon the calculated extinctioncoefficient, ε₂₈₀=29,870 M⁻¹ cm⁻¹ in a final buffer concentration of 6 Mguanidinium hydrochloride, 20 mM phosphate buffer, pH 6.5 (Gill and vonHippel, 1989).

Example 2 96-Well Plate Screen for Methionine-γ-Lyase Activity andRanking Clones

Both MGL and CGL produce 2-ketobutanoic acid from their respectivesubstrates. A colorimetric assay for the detection of α-keto acids3-methylbenzothiazolin-2-one hydrazone (MBTH) (Takakura et al., 2004)was scaled to a 96-well plate format for screening small libraries andfor ranking clones with the greatest METase (methionine-γ-lyase)activity.

Single colonies encoding mutant enzymes displaying activity in the assaydescribed in the preceding paragraph are picked into microwells in96-well plates containing 75 μL of TB media/well and 50 μg/ml kanamycin.Cells are grown at 37° C. on a plate shaker until reaching an OD₆₀₀ of0.8-1. After cooling to 25° C., an additional 75 μL of media/wellcontaining 50 μg/ml kanamycin and 0.5 mM IPTG is added. Expression isperformed at 25° C. with shaking for 2 hrs, following which 100 μL ofculture/well is transferred to a 96 well assay plate. The assay platesare then centrifuged to pellet the cells, the media is removed, and thecells are lysed by addition of 50 μL/well of B-PER protein extractionreagent (Pierce). After clearing by centrifugation, the lysate isincubated with 5 mM L-Met at 37° C. for 10-12 hrs. The reaction is thenderivatized by addition of 3 parts of 0.03% MBTH solution in 1 M sodiumacetate pH 5. The plates are heated at 50° C. for 40 min and aftercooling are read at 320 nm in a microtiter plate reader.

Example 3 Genetic Selection for Methionine-γ-Lyase Activity

α-ketobutyrate is produced by the action of threonine deaminase (encodedby ilvA) in E. coli as the first enzyme in the isoleucine biosyntheticpathway. Almost 40 years ago, Grimminger and Feldner identifiedthreonine deaminase mutants that were isoleucine auxotrophs (Grimmingerand Feldner 1974) and more recently a threonine deaminase point mutantwas found in a common expression strain BLR(DE3) that reportedly onlyhas <5% of normal growth capacity when isoleucine is omitted from media(Goyer et al., 2007). Expression of methioninase, which producesα-ketobutyrate, was shown to rescue the growth of ilvA mutants and thusallow colony formation in minimal media. However, the threoninedeaminase gene in BLR(DE3) is a point mutant, making reversion adistinct likelihood especially in very large libraries. Threoninedeaminase is the distal gene in the isoleucine biosynthetic operonilvGMEDA and is expressed as part of the operon or independently from aninternal promoter (Lopes and Lawther 1989).

In order to avoid undesired effects on the expression of other Ile genesinternal fragments within the 1545 bp ilvA gene of E. coli strain BL21were deleted using E. coli strain JW3745-2 (Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), λ-, rph-1, ΔilvA723::kan, Δ(rhaD-rhaB)568, hsdR514)obtained from the Yale E. coli Genetic Stock Center (New Haven, Conn.)through P1 transduction and curing of the kanamycin resistance marker byusing the FLP recombinase plasmid pCP20 as described elsewhere (Datsenkoand Wanner 2000). It has also been noted that L-cystathionine andL-homocysteine are intermediates in the E. coli methionine biosyntheticpathway (FIG. 11). L-cystathionine and L-homocysteine are substrates ofcystathionine-γ-lyase that result in production of α-ketobutyrate andallow complementation of the isoleucine biosynthetic pathway. Thereforethe gene metA (encoding homoserine-O-succinyltransferase) was knockedout using an E. coli strain JW3973-1 (Δ(araD-araB)567,ΔlacZ4787(::rrnB-3), λ-, rph-1, Δ(rhaD-rhaB)568, ΔmetA780::kan, hsdR514)obtained from the Yale E. coli Genetic Stock Center (New Haven, Conn.)through P1 transduction and curing of the kanamycin resistance marker byusing the FLP recombinase plasmid pCP20 as described elsewhere (Datsenkoand Wanner 2000). E. coli strain BL21 (DE3) (F-ompT gal dcm lon hsdSB(rB-mB-) λ(DE3 [lacI lacUV5-T7 gene 1 ind1 sam7 nin5]) was used as arecipient strain, resulting in an E. coli strain BL21(DE3) (ΔilvA,ΔmetA) which is auxotrophic for both L-isoleucine and L-methionine.

Thus E. coli BL21(DE3) (AilvA, AmetA)-harboring plasmids containing thegenes for a methionine-γ-lyase, an engineered cystathionine-γ-lyase, cangrow on M9-minimal media plates supplemented with L-methionine, but thesame strain having only wild-type cystathionine-γ-lyase and an emptyplasmid cannot (FIG. 12). Very large libraries of recombinantlyexpressed engineered cystathionine-γ-lyase variants that havemethionine-γ-lyase activity can be thus be rapidly selected for againsta background of inactive clones or clones with cystathionase activity.Larger colonies and those appearing more quickly are indicative of moreactive enzymes.

Example 4 Effect of Mutagenesis Upon Residues E59, R119, and E339 ofhCGL

Structural analysis indicated that residues E59, R119, and E339 arelikely involved in the recognition of hCGL for it substrateL-cystathionine. NNS codon saturation libraies were constructed at thesesites and screened using the following mutagenic primers: (E59) Forward‘5-ggccagcatagcggttttNNStatagccgtagcggc (SEQ ID NO:2), Reverse‘5-GCCGCTACGGCTATASNNAAAACCGCTATGCTGGCC (SEQ ID NO:3), (R119) Forward‘5-gtatggtgggaccaatNNStatttccgtcaggtggcg (SEQ ID NO:4), Reverse‘5-CGCCACCTGACGGAAATASNNATTGGTCCCACCATAC (SEQ ID NO:5), (E339) Forward‘5-ctgaaactgtttaccctggcaNNSagcttgggcggctttg (SEQ ID NO:6), and Reverse‘5-CAAAGCCGCCCAAGCTSNNTGCCAGGGTAAACAGTTTCAG (SEQ ID NO:7), using thehCGL gene as template DNA and specific end primers; forward‘5-gatataccATGGGAGGCCATCACCACCATCATCATGGCGGGCAGGAAAAGGATGCG (SEQ IDNO:8) and reverse‘5-CTCGAATTCTCAACTGTGGCTTCCCGATGGGGGATGGGCCGCTTTCAGCGCCTGATC C (SEQ IDNO:9). The PCR product was digested with NcoI and EcoRI and ligated intopET28a vector with T4 DNA ligase. The resulting ligations weretransformed directly into E. coli (BL21) and plated on LB-kanamycinplates for subsequent screening as described in Example 2. Two timesmore colonies than the theoretical diversity of the libraries werescreened. Clones displaying activity were isolated and the sequence ofthe hCGL gene was determined to identify the mutations.

The enzyme variants were purified to greater than 95% homogeneity asassessed by SDS-PAGE. Incubation with PLP was shown to enhance thespecific activity presumably because the E. coli cells used forexpression do not produce sufficient PLP for all the hCGL produced. Oncethe enzyme had been loaded with PLP it was stable with no loss of thecofactor upon storage.

Example 5 Characterization of Human Cystathionine-γ-Lyase Variants

Two hCGL variants identified from the screen as having the highestcatalytic activity were found to have the following mutations: E59N orV, R119L, and E339V, as well as an N-terminal His6 tag addition. Thesevariants were called hCGL-NLV (SEQ ID NO:10), and hCGL-VLV (SEQ IDNO:11) respectively. Both of these variants were characterizedkinetically for their ability to degrade L-Met in a 100 mM PBS buffer atpH 7.3 and 37° C. using a 1 ml scale MBTH assay similar to thatdescribed in Example 2. The Ellman's reagent (DTNB) to detect release ofmethane thiol from hCGL variant catalysis of L-Met, resulting in afacile continuous assay. Both hCGL-NLV, and hCGL-VLV had a k_(cat)/K_(M)of ˜1×10⁴ s⁻¹ M⁻¹ for L-Met. The parent enzyme native hCGL was not ableto degrade L-Met within the detection limits of these assays.

The serum stability of methionases was tested by incubating of theenzyme in pooled human serum at 37° C. and at a final concentration of10 μM. At different time points, aliquots were withdrawn and tested foractivity. After plotting the data the MGL from P. putida was found tohave an apparent T_(0.5) of 2±0.1 hrs. Human CGL showed an apparentT_(0.5) of 16±0.8 hrs. Surprisingly, hCGL-NLV and CGL-VLV weredramatically more stable with the former showing an apparent T_(0.5) of95±3 hrs, and hCGL-VLV with an apparent T_(0.5) of 260±18 hrs (FIG. 2).

The thermal stability of the methioninase enzymes was also evaluated bycircular dichroism spectroscopy (CD). Human CGL and variant hCGL-NLV hadapparent T_(M) values of ˜68° C., hCGL-VLV was slightly more stable withan apparent T_(M) of ˜71° C. (FIG. 3). The similar T_(M) values of hCGL,hCGL-NLV, and hCGL-VLV suggests that the extended serum stability is dueto the PLP cofactor being preferentially retained in the active-site ofhCGL-NLV, and hCGL-VLV.

Example 6 Cytotoxicity of Human Methionine-γ-Lyase Against Neuroblastoma

The in vitro cytoxicity of hCGL and pMGL with the neuroblastoma cellline Lan-1 was evaluated. LAn-1 cells were seeded at 7000 cells/well andincubated with varying concentrations of pMGL or hCGL-NLV. After 3-5days exposure proliferation was measured using WST-8 previously and thedata plotted to calculate apparent ICso values (Hu and Cheung 2009).Analysis of the resulting data (FIG. 6) yielded an apparent ICso valueof 0.34 U/ml (˜0.6 μM) for pMGL treated cells and an apparent ICso valueof 0.15 U/ml (˜0.3 μM) cells treated hCGL-NLV.

Example 7 Cytoxicity of PEGylated Human Methionine-γ-Lyase Against aPanel of Neuroblastoma Cell Lines

In vitro proliferation of NB cell lines (BE(1)N, BE(2)N, BE(2)S, BE(2)C,SK-N-LD, NMB-7, SH-EP-1, IMR32, CHP-212, SKN-MM, LAN-1, LAI-66N,LAI-55N, LAI-5S) was assayed in 96-well plates (BD Biosciences, Bedford,Mass.) with varying concentrations of PEG-hMGL or PEG-hCGL-NLV.

Twenty-four hours after seeding at a density of 3000-6000 cells/well,PEG-hCGL-NLV and pMGL were added. After 3 days of culture, PEG-hMGL andpMGL were removed, fresh medium added, and cells were incubated foranother 36 hr. WST8 (Dojindo Molecular Technologies, MA) was added toculture wells at volume ratio of 1:10. After 4-6 hr of reaction, opticaldensity (OD) was read at 450 nm. Cell proliferation was calculated asfollows: % Cell Growth=(OD₄₅₀ of experimental well−OD₄₅₀ of medium-onlywell)/(OD₄₅₀ of control well−OD₄₅₀ of medium-only well)×100%. IC₅₀ (halfmaximal inhibitory drug concentration) was calculated using SigmaPlot8.0 (Systat Software, Inc., San Jose, Calif.). PEG-hCGL-NLV showedcytoxicity against all cell lines tested with IC50 values ranging from0.175-0.039 U similar to the pseudomonas MGL that had IC50 valuesranging from 0.174-0.042 U (FIG. 9).

Example 8 Pharmacological Preparation of Human Cystathionine-γ-LyaseVariants

hCGL-NLV was purified as described in Example 1 with one exception:after binding to the IMAC column, the protein is washed with extensively(90-100 column volumes) with an IMAC buffer containing 0.1% Triton 114in the sample. 10-20 column volumes of IMAC buffer, and then eluted withan IMAC elution buffer (50 mM NaPO₄/250 mM imidazole/300 mM NaCl, pH 8).Wash with Triton 114 was employed to reduce endotoxin(lipopolysaccharide) contamination. The purified protein was subjectedto buffer exchange into a 100 mM NaPO₄, buffer at pH 8.3 using a 10,000MWCO filtration device (Amicon). Subsequently, PLP was added at aconcentration of 10 mM and the protein was incubated for 1 hr at 25° C.Methoxy PEG Succinimidyl Carboxymethyl Ester 5000 MW (JenKem Technology)was then added to hCGL-NLV at a 80:1 molar ratio and allowed to reactfor 1 hr at 25° C. under constant stirring. The resulting mixture wasextensively buffer exchanged (PBS with 10% glycerol) using a 100,000MWCO filtration device (Amicon), and sterilized with a 0.2 micronsyringe filter (VWR). All pegylated enzymes were analyzed forlipopolysaccharide (LPS) content using a Limulus Amebocyte Lysate (LAL)kit (Cape Cod Incorporated).

Analysis by SDS-PAGE and size exclusion chromatography (FIGS. 4-5) showsthat an 80 fold molar excess of PEG MW5000 greatly increases thehydrodynamic radius of hCGL variants with MGL activity from ˜220 kDa forthe unpegylated tetramer to an estimated 1,340 kDa for PEGylated hCGLvariants. Pegylated human methionase was found to have nearly identicalkinetic activity and in vitro serum stability as compared to theun-PEGylated enzyme.

Example 9 Pharmacodymanic Analysis of PEG MW 5000 hCGL-NLV in Mice

The in vivo half-life of PEGylated hCGL variants was determined in mice(balb/c) (n=5) following tail vein injection with 50 U of PEG-hCGL-NLV.Blood samples were withdrawn at different times and hCGL-NLV activitywas determined as above. The activity in blood withdrawn at t=1 hr wasset at 100%. An exponential fit to the data revealed a T½ of 28±4 hrs(FIG. 7), a 14-fold improvement compared to PEG-pMGL.

Example 10 Methionine Depletion in Mouse Plasma

Mice (n=5) fed on a Methionine(−) Homocystine(−) Choline(−) (Met(−)Hcyss(−)Chl(−)) diet prior to treatment were dosed with 200 U ofPEG-hCGL-NLV by tail vein injection. Plasma samples were analyzed forL-Met levels by HPLC basically as described elsewhere (Sun, et al.2005). Blood methionine levels decreased from 124±37 μM prior totreatment to a minimum of 3.9±0.7 μM at 8 hrs and were kept low for over24 hrs (FIG. 8).

Example 11 Effect of PEG-hCGL-NLV on LAN-1 Tumor Xenografts

A schedule for PEG-hCGL-NLV administration was devised to minimizeweight loss and maximize pharmacokinetics of PEG-hCGL-NLV. (The onlydose limiting toxicity in vivo is weight loss after methioninedeprivation.) When athymic mice xenografted with LAN-1 were treated with100 Units PEG-hCGL-NLV i.v. thrice a week for 4 weeks, a 15-20%reversible weight loss was observed. 24 hr after the third PEG-hCGL-NLVinjection during the last (4^(th)) week of treatment, plasma methionineconcentrations were 5.8±2.1 μM among PEG-hCGL-NLV-treated mice fed withMet(−)Hcyss(−)Chl(−) diet, in contrast to 124±37 μM before treatment(n=10). Mice fed Met(−)Hcyss(−)Chl(−) diet had 10-15% reversible weightloss and the plasma methionine concentration was 13.2±4.5 μM while onMet(−)Hcyss(−)Chl(−) diet. When 100 Units PEG-hCGL-NLV treatment wascombined with Met(−)Hcyss(−)Chl(−) diet, significant anti-tumor effectagainst LAN-1 xenografts (p<0.01) was observed when compared to the notreatment group or the group receiving only Met(−)Hcyss(−)Chl(−) diet(FIG. 10).

Example 12 Engineering of Primate Methionine-γ-Lyases

The sequences of CGLs from primate species such as chimpanzees (Pantroglodytes), orangutans (Pongo abelii), and macaques (Macacafascicularis) are respectively about 99, 96, and 95% identical in aminoacid composition to human CGL. Primate CGL enzymes with mutationsconferring Methionine-γ-lyase activity are constructed using standardmutagenesis techniques as described in Example 4. The resulting genesare cloned into pET28a Non-human primate hGCL with L-methionase activityare then expressed and purified asdescribed above. Primate CGLsengineered with amino acid positions corresponding to N, or V59, L119,and V339 degrade L-Met with k_(cat)/K_(M) values of at least 1×10²s⁻¹M⁻¹.

Examples of amino acid sequences of engineered primate CGLs with MGLactivity are disclosed as below:

Pongo abelii CGL-NLV (SEQ ID NO:12, with V59N, R119L, and E339Vsubstitutions and addition of an N-terminal His6 tag on the nativesequence having Genbank ID NP_001124635.1 (i.e., SEQ ID NO:18)), Pongoabelii CGL-VLV (SEQ ID NO:13, with R119L and E339V substitutions andaddition of an N-terminal His6 tag on the native sequence having GenbankID NP_001124635.1);

Macaca fascicularis CGL-NLV (SEQ ID NO:14, with E59N, R119L, and E339Vsubstitutions and addition of an N-terminal His6 tag on the nativesequence having Genbank ID AAW71993.1 (i.e., SEQ ID NO:19)), Macacafascicularis CGL-VLV (SEQ ID NO:15, with E59V, R119L, and E339Vsubstitutions and addition of an N-terminal His6 tag on the nativesequence having Genbank ID AAW71993.1);

Pan Troglodytes CGL-NLV (SEQ ID NO:16, with E59N, R119L, and E339Vsubstitutions and addition of an N-terminal His6 tag on the nativesequence having Genbank ID XP_513486.2 (i.e., SEQ ID NO:20)), and PanTroglodytes CGL-VLV (SEQ ID NO:17, with E59V, R119L, and E339Vsubstitutions and addition of an N-terminal His6 tag on the nativesequence having Genbank ID XP_513486.2).

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1.-32. (canceled)
 33. A method of identifying a primate cystathionine gamma-lyase variant having L-methionine degrading activity, comprising: a) expressing a population of primate cystathionine gamma-lyase variants in host cells, wherein the variants of the population comprise at least one amino acid substitution as compared to a native primate cystathionine gamma-lyase; and b) identifying a host cell expressing a primate cystathionine gamma-lyase variant having L-methionine degrading activity.
 34. The method of claim 33, wherein the host cells are bacterial host cells.
 35. The method of claim 34, wherein the bacterial host cells are E. coli host cells.
 36. The method of claim 35, wherein the E. coli host cells have deletions of genes ilvA and metA.
 37. The method of claim 33, wherein said variant is identified by identifying a host cell having a higher growth rate in a minimal medium supplemented with L-methionine as compared to cells expressing the native primate cystathionine gamma-lyase in otherwise similar conditions. 